Diaphorase is an enzyme which reversibly catalyzes the reaction of converting NAD(P)+ to NAD(P)H and transfers its electrons to a variety of Redox dyes, e.g. resazurin also known as Alamar Blue. The enzyme from Clostridium kluyveri could utilize both NAD(H) and NADP(H) with similar efficiency; thus is an attractive coupling enzyme for detection of oxidoreductase- and dehydrogenase-based more ..

Diaphorase is an enzyme which reversibly catalyzes the reaction of converting NAD(P)+ to NAD(P)H and transfers its electrons to a variety of Redox dyes, e.g. resazurin also known as Alamar Blue. The enzyme from Clostridium kluyveri could utilize both NAD(H) and NADP(H) with similar efficiency; thus is an attractive coupling enzyme for detection of oxidoreductase- and dehydrogenase-based reactions. For example, diaphorase from Clostridium kluyveri was utilized in the screening of phosphomannose isomerase (PMI) paired with resazurin (AID 1209). In this fluorogenic assay the NADPH produced in the reaction reduces resazurin to form resorufin with a concomitant increase in red fluorescence.

The purpose of the current assay assay is to identify inhibitors of diaphorase. It is expected that strong inhibitors of diaphorase would appear as inhibitors in the PMI assay. Thus, diaphorase is a counter-screen for the PMI and other diaphorase-based assays allowing identification of the artifacts of the coupled assay detection system. The concentration of NADH substrate of diaphorase was kept at its Km value to resemble the conditions of the coupled reactions.

In addition, compounds that have strong oxidative properties are also expected to suppress the resorufin formation and appear as inhibitors of diaphorase based reactions. Thus, the current assay could be utilized for library characterization in respect to redox-active compounds.

1) 2 uL of Substrate working solution was added to columns 3-48 of a Costar 1536-well black plate (cat #3724) using a Thermo Multidrop Combi dispenser2) 2 ul of Substrate working solution without NADH was to added to columns 1 and 2 (positive control) of a Costar 1536-well black plate (cat #3724) using a Thermo Multidrop Combi dispenser 3) 40 nL of 100% DMSO was added to columns 1-4 using a HighRes biosolutions pintool and V&P Scientific pins3) 40 nL of 2 mM compounds in 100% DMSO were dispensed in columns 5-48 using a HighRes biosolutions pintool and V&P Scientific pins4) 2 uL of Enzyme working solution was added to the whole plate using a Thermo Multidrop Combi dispenser. 5) Plates were incubated at room temperature for 16 min. 6) After 16 minutes the plates were read on a ViewLux plate reader (Perkin Elmer), Ex544, Em590.7) The screening was performed using a HighRes biosolution fully integrated HTS POD-based system8) Data analysis was performed using CBIS software (ChemInnovation, Inc).

Diaphorase enzyme used in this assay is from Clostridium kluyveri and its protein/nucleotide sequence is not available in GeneBank. Sequence of human counterpart of this enzyme is cited in the Target Data reference.

Compounds with greater than 50% inhibition at 20 uM concentration are defined as actives of the primary screening. The primary screening actives proceed to the dose-response confirmation stage.

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the diaphorase assay is described below.

Activity ScoringActivity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the diaphorase assay is as follows: 1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % displacement in the assay demonstrated by a compound at 20 uM concentration:a. If primary % inhibition is less than 0%, then the assigned score is 0b. If primary % inhibition is greater than 100%, then the assigned score is 40c. If primary % inhibition is between 0% and 100%, then the calculated score is (% Inhibition)*0.42) Second tier (41-80 range) is reserved for dose-response confirmation data 3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues